Eszter Molnar

Background: BRAF is key component in the growth factor signaling cascade that is often impaired in tumors. Although oncogenic BRAF mutation is considered to be the driver mutation in a variety of malignancies, the response to RAS/RAF/PI3K signaling network inhibition is not uniform among BRAF mutant tumors. Accordingly, we investigated the in vitro effect of RAS/RAF/PI3K signaling network inhibitors (panRAF, mutant BRAF specific, MEK and PI3K/mTOR inhibitors) in relation with BRAF concomitant RAS or PTEN/PI3K mutations in a panel of human cancer cell lines. Methods: Altogether 12 human cancer cell lines were investigated in vitro to assess the effect of inhibitors acting at the different levels of the RAS/RAF signaling network. The seven melanoma, two colon, two lung and one breast cancer cell lines were divided into 3 groups according to their mutational status: BRAF-mutant with no PTEN/PI3K/RAS mutations (A375, WM35, SK-MEL-28, CRL5885), BRAF + PTEN-PI3K (A2058, WM239, HT29, SW1417) and BRAF + RAS double mutant (WM3629, WM3670, MDA-MB-231, CRL5922). Effect of inhibitors on proliferation, clonogenic potential and migration were investigated SRB and clonogenic assays and videomicroscopy measurements, respectively. Both single agent and combination treatments were applied. Results: In single agent treatments panRAF and PI3K/mTOR inhibitors did not show mutation dependent differences in proliferation, however, mutant-BRAF and MEK specific inhibitors were more effective in cells without concomitant PTEN/PI3K or RAS mutations. Interestingly, not only the combination of BRAF and MEK inhibitors but the combination of PI3K/mTOR and MEK inhibition showed additive inhibitory effect on proliferation in all subgroups. Importantly, a distinct pattern emerged for the mutational subgroup specific migration inhibitory effects in comparison to the inhibition of proliferation. Conclusion: BRAF mutant cells carrying various concomitant PTEN/PI3K/RAS alterations display distinct response to RAS/RAF/PI3K network inhibition in terms of proliferation as well as migration. Our data suggests that combination treatments acting concurrently on different targets of the RAS/RAF/PI3K network can be more effective as single therapies. Citation Format: Tamas M. Garay, Eszter Molnar, Dominika Rittler, Walter Berger, Balazs Döme, Jozsef Timar, Balazs Hegedus. Oncogenic BRAF and concomitant RAS or PTEN/PI3K mutations determine together the response to RAS/RAF/PI3K signaling network inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3831.

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4b expression in gastric, colon and breast cancer cells. In the present study we treated BRAF mutant and BRAF wild type melanoma cells with HDAC inhibitors (SAHA (suberanilohydroxamic acid) or valproic acid) and tested the changes in abundance, localization and function of PMCA4b. Expression levels of the PMCA proteins were analyzed by qRT-PCR and Western Blotting. Subcellular localization of the PMCAs and the effects of treatments on cytosolic Ca2+ signaling were analyzed by confocal microscopy. We found that treatment with the HDAC inhibitors increased the level of PMCA4b expression at both mRNA and protein levels in both BRAF wild type and BRAF mutant cell types. The increased PMCA4b level was coupled with enhanced plasma membrane localization and with a faster Ca2+ clearance after stimulation. Our results show that in melanoma cells the expression of PMCA4b is under epigenetic control and HDAC inhibitors efficiently enhanced PMCA4b expression independent of the BRAF status of cells. Citation Format: Luca Hegedus, Tamas Garay, Eszter Molnar, Karolina Varga, Rita Padanyi, Katalin Paszty, Balazs Hegedus, Mathias Wolf, Michael Grusch, Eniko Kallay, Agnes Enyedi. Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1883.